This invention relates to a transgenic animal model of Alzheimer""s disease and related neurological disorders in which the animal harbors a transgene encoding a protease inhibitor such as antichymotrypsin (ACT) protein. The invention further relates to transgenic animal models of Alzheimer""s disease and related neurological disorders in which the animal harbors a transgene encoding a protease inhibitor such as antichymotrypsin (ACT) protein and one or more further transgenes affecting said neurological disorders. The invention further relates to cells comprising a transgene encoding an antichymotrypsin (ACT) protein. The invention also relates to drug screening assays using the invented transgenic cells or transgenic animals and progeny thereof.
Biochemical, genetic, and epidemiological evidence indicates that inflammation is an essential part of the pathogenesis of Alzheimer""s disease. For example, several acute phase/inflammatory molecules in the brain, specifically antichymotrypsin (ACT) and apolipoprotein E (apoE) can promote the formation of the neurotoxic amyloid deposits that are the main pathological hallmark of the disease. For further details and background information on Alzheimer""s disease and related neurological diseases see, for example, U.S. Pat. Nos. 5,297,562; 6,043,283; 5,986,054; 5,981,208; 5,958,883; 5,854,215; 5,849,560; 5,830,670; 5,817,626; 5,773,220; 5,753,624; 5,731,284; 5,705,401; 5,571,671; 5,535,760; 5,506,097; 5,449,604; 5,434,170; 5,429,947; and 5,276,059 as incorporated herein by way of reference.
Amyloid plaque formation is found in a number of diseases including Alzheimer""s Disease (AD), scrapie, bovine spongiform encephalopathy, Gerstmann-Straussler Syndrome and related transmissible spongiform encephalopathies (TSEs). These amyloid plaques comprise protein molecules bound together in a fibrinous matrix. Other disorders, such as Creutzfeldt-Jakob""s disease, are characterized by the accumulation of amyloidogenic protein without deposition of amyloid plaques. Together these groups of conditions are referred to hereinafter as xe2x80x9cAmyloidogenic Diseases.xe2x80x9d The present inventors were the first to discover that the acute phase protein, alpha1-antichymotrypsin (ACT) is a structural component of Alzheimer amyloid deposits. In vitro and in vivo studies have shown that the increased ACT expression in Alzheimer""s disease is induced in astrocytes by the inflammatory cytokine IL-1 released from reactive microglial cells in the regions of amyloid deposition (Das, S. and Potter, H. xe2x80x9cExpression of the Alzheimer amyloid-promoting factor antichymotrypsin is induced in human astrocytes by IL-1xe2x80x9d Neuron 14:447-465,1995).
ACT is an inhibitor of chymotrypsin-like serine proteases and is normally produced in the liver as part of the body""s xe2x80x9cacute phase responsexe2x80x9d to inflammation. An important function of the acute phase response is to increase the general level of anti-protease activity in the body so as to reduce the potential damage that inflammation-associated proteases can inflict on normal serum proteins and healthy tissue outside of the immediate area of inflammation. The finding that ACT is overexpressed in astrocytes in affected areas of the Alzheimer brain provides the first clear indication that inflammation and an acute phase response in the brain are part of the disease. Other, independent, biochemical and epidemiological studies have confirmed the likelihood that these processes indeed play important roles in the pathogenesis of Alzheimer""s disease. The important conclusion is that Axcex2 does not act alone to cause Alzheimer""s disease, but acts in concert with an inflammatory cascade, whose products are required for efficient amyloid formation.
The findings that ACT, together with the Axcex2 peptide, is an integral component of the Alzheimer amyloid filaments, and that the mature amyloid deposits are restricted to the same brain regions in which ACT is overproduced, led to the proposal that ACT contributes directly to amyloid formation (Abraham, C. R., Selkoe, D. J. and Potter, H. xe2x80x9cImmunohistochemical identification of the serine protease inhibitor xcex11-antichymotrypsin in the brain amyloid deposits of Alzheimer""s diseasexe2x80x9d Cell 52:487-501,1990). When apolipoprotein E was found to be also present in Alzheimer amyloid, suggesting a similar role, the term xe2x80x9cpathological chaperonexe2x80x9d was coined to describe the potential function of these two, and possibly other proteins, in amyloid formation (Wisniewski, T. and Frangione, B. xe2x80x9cApolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloidxe2x80x9d Neurosci. Lett. 135:235-238, 1992). Indeed, when ACT or apoE are added to preparations of synthetic Axcex2 peptide in vitro, they promote the polymerization of Axcex2 into amyloid filaments (Ma, J., Yee, A. Brewer, H. B. Jr. and Das, J. and Potter, H. xe2x80x9cAmyloid-associated proteins xcex11-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer""s xcex2-protein into filamentxe2x80x9d Nature 372:92-94, 1994; Wisniewski, T., Castano, E. M., Golabek, A., Vogel, T. and Frangione, B. xe2x80x9cAcceleration of Alzheimer""s fibril formation by apolipoprotein E in vitroxe2x80x9d Am. J. Pathol. 145:1030-1035, 1994; Sanan, D. A., Weisgraber, K. H., Russell, S. J., Mahley, R. W., Huang, D., Saunders, A., Schmechel, D., Wisniewski, T., Frangione, B., Roses, A. D. and Strittmatter, W. J. xe2x80x9cApolipoprotein E associates with beta amyloid peptide of Alzheimer""s disease to form novel monofibrils. Isoform apoE4 associates more efficiently than apoE3xe2x80x9d J. Clin. Invest. 94:860-869, 1994). ApoE4, the isoform of apoE, identified by epidemiological studies as a strong risk factor for inherited Alzheimer""s disease (Strittmatter, W. et. al. xe2x80x9cApolipoprotein E: high avidity binding to xcex2-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer""s diseasexe2x80x9d Proc Natl Acad Sci USA 90, 1977-1981, 1993; Corder, E. et. al. xe2x80x9cGene dose of apolipoprotein E type 4 allele and the risk of Alzheimer""s disease in late onset familiesxe2x80x9d Science 261, 921-923, 1993), is a much more active amyloid promoting factor than the non-pathogenic apoE3 or apoE2 isoforms. Furthermore, the greater number and length of the filaments formed under the promoting effect of ACT and apoE4 show increased toxicity to human cortical neurons in culture (Ma, J., Brewer, H. B. Jr., and Potter, H. xe2x80x9cAlzheimer A beta neurotoxicity: promotion by antichymotrypsin. ApoE4; inhibition by A beta-related peptides.xe2x80x9d Neurobiol Aging 17:773-78, 1996). Together, these results support the hypothesis that Alzheimer""s disease involves an inflammation-like reaction and a consequent acute phase response in the brain that is essential for the development of mature amyloid neuropathology and neuronal cell death.
Since demonstration that ACT and apoE, especially apoE4, are amyloid promoters in vitro, many other proteins have been tested for their possible effect on Axcex2 polymerization, such as amyloid P component, acetylcholinesterase and complement factors (for review see Nilsson, L., Rogers, J. and Potter, H. xe2x80x9cThe essential role of inflammation and induced gene expression in the pathogenic pathway of Alzheimer""s diseasexe2x80x9d Front. Biosci. 16:426-446, 1998). ACT and apoE are the only such molecules for which genetic studies support their involvement in the Alzheimer pathogenic pathway. For example, one of the greatest genetic risk factors for developing Alzheimer""s disease is the inheritance of one, or worse, two copies of the apoE4 allele. Furthermore, inheritance of apoE leads to increased numbers of amyloid deposits in both Alzheimer patients and normal aged individuals, suggesting that apoE plays a direct role in amyloid formation, rather than, for instance increasing cells"" sensitivity to neurotoxicity.
Genetic support for the involvement of ACT in Alzheimer""s disease has been not so obvious as for apoE4. In one study, the inheritance of a specific isoform of ACT (an alanine instead of a threonine in the signal peptide) correlated with a 8-fold increased risk of developing Alzheimer""s disease in apoE4 carriers (Kamboh, M. I., Sanghera, D. K., Ferrell, R. E. and DeKosky, S. T. xe2x80x9cApoE4-associated Alzheimer""s disease risk is modified by xcex11-antichymotrypsin polymorphismxe2x80x9d Nature Genet. 10:486-488, 1995). Since this single amino acid change does not affect the secreted protein itself and therefore cannot alter its affinity for the Axcex2 peptide or its ability to promote Axcex2 polymerization, it is possible that the effect is on the synthesis and secretion of the ACT protein. This result has been confirmed and modified by several others (Thome, J., Baumer, A., Kornhuber, J., Rosler, M., and Riederer, P. xe2x80x9cAlpha-1-antichymotrypsin bi-allele polymorphism, apolipoprotein E tri-allele polymorphism and genetic risk of Alzheimer""s syndromexe2x80x9d J. Neural Trans. 10:207-212, 1995; Morgan et al.; 1996; Talbot, C., Houlden, H., Craddock, N., Crook, R., Hutton, M., Lendon, C., Prihar, G., Morris, J. C., Hardy, J. and Goate, A. xe2x80x9cPolymorphisms in AACT gene may lower age of onset of Alzheimer""s diseasexe2x80x9d Neuroreport. 7:534-536, 1996). Most recently, a study showed that inheritance of the ACT-A allele (independent of apoE alleles) was highly correlated to the extent of amyloid angiopathy in the brain (Yamada, M., Sodeyma, N., Itoh, Y., Suematsu, N., Otomo, E., Matsushita, M. and Mizusawa, H. xe2x80x9cAssociation of the alpha1-antichymotrypsin polymorphism with cerebral amyloid angiopathyxe2x80x9d Ann. Neurol. 44:129-131, 1998).
Although the pathological evidence (such as the overexpression of ACT in affected areas of AD brain and the increased amyloid load in apoE4 and ACT-A carriers) and much of the biochemical evidence, has pointed to such proteins being amyloid promoters, it is also possible from the genetic data alone that, for instance, apoE can be an amyloid inhibitor with apoE4 being a less effective inhibitor than apoE3. Indeed, there exist in vitro studies in which apoE or ACT appeared to inhibit Axcex2 polymerization (see, for example, Evans, K. C., Berger, E. P., Cho, C-G, Weisgraber, K. H. and Lansbury, P. T. Jr. xe2x80x9cApolipoprotein E is a kinetic but not a thermodynamic inhibitor of amyloid formation: implications for the pathogenesis and treatment of Alzheimer""s diseasexe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 92:763-767, 1995; Eriksson, S., Janciauskiene, S. and Lannfelt, L. xe2x80x9cxcex11-antichymotrypsin regulates Alzheimer xcex2-amyloid peptide fibril formationxe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 92:2313-2317, 1995; Fraser, P. E., Nguyen, J. T., McLachlen, D. R., Abraham, C. R. and Kirschner, D. A. xe2x80x9cxcex11-antichymotrypsin binding to Alzheimer Axcex2 peptides is sequence specific and induces fibril disaggregation in vitroxe2x80x9d J. Neurochem. 61:298-305, 1994). The further confusion for the role apoe has recently been provided by a series of in vivo experiments that show it to be an amyloid promoter (Bales, K. R.; Verina, T., Dodel, R. C., Du, Y., Altstiel, L., Bender, M., Hyslop, P., Johnstone, E. M., Little, S. P., Cummins, D. J., Piccardo, P., Ghetti, B. and Paul, S. M. xe2x80x9cLack of apoliprotein E dramatically reduces amyloid xcex2-peptide depositionxe2x80x9d Nature Genet. 17:263-264, 1997). Specifically, a set of mouse strains were developed that expressed transgenic human APP but which had their apoE gene either half (heterozygous) or completely (homozygous) knocked out. The animals showed a variable amount and speed of amyloid deposition that was absolutely dose-dependent on the number of copies of the apoE gene. If there was no apoE, mature, filamentous amyloid did not form in animals up to two years of age, compared to massive amyloid deposition by 7 months in the presence of the normal two copies of the apoE gene (Bales et al., 1997, supra). One copy of apoE gave intermediate results. It therefore appears that human Axcex2, by itself, is incapable of forming amyloid in the mouse without the promoting effect of apoE. Therefore, the current state of the art is still contradictory as there are studies supporting two opposite roles for ACT and other amyloid plaque-associated factors.
If APP mice lacking apoE fail to form amyloid filaments, one might ask whether ACT still has a role to play in amyloid formation, or whether apoE is sufficient. However, such a study is complicated by the fact that mice do not have an ACT gene per se. The closest mouse homologue is contrapsin which is quite different from ACT. Contrapsin does not bind Axcex2, and is not expressed in the same way as ACT. Therefore, apoE may be the only, or most important amyloid promoter in mice, but ACT remains a candidate for a similar function in humans. It would be desirable, therefore, in the field of the present invention, to study the amyloid-promoting and/or suppressing effect of ACT, under in vitro conditions to correctly identify its role as an amyloid promoter/inhibitor.
The defining pathological characteristics of Alzheimer""s disease are plaques, tangles, inflammation, and neuronal degeneration. However, very little is known about the mechanism by which tangles and their component paired helical filaments form and the precise relationship between neuronal degeneration and the other pathological features of AD. It is clear that post-translationally modified microtubule associated protein tau is the major PHF protein, but what induces the modifications such as hyperphosphorylation of certain amino acids in AD is unknown. It has been particularly irksome that the best transgenic models of AD expressing mutant human genes that cause inherited AD form plaques and exhibit inflammation but do not develop tangles or neuronal cell death. It would therefore be advantageous for an animal model of Alzheimer""s disease to exhibit Tau protein pathology found in the human disease, including hyperphosporylated Tau, paired helical filaments, neurofibrillary tangles and neuronal degeneration.
Several prior unsuccessful attempts have been made to generate transgenic mice expressing ACT. In a first attempt in 1989 by the present inventors, the ACT coding region was linked to the SV40 promoter for driving expression in all tissues, since no astrocyte-specific promoters had yet been characterized. Unfortunately, general expression of this protease inhibitor resulted in the animals dying within a week of birth.
A second attempt to generate ACT mice was performed in which the glial fibrillary acidic protein (GFAP) promoter was used. However, no ACT protein expression occurred in the brains of the animals, even after a stab wound was used to induce gliosis (Mucke et al. 2000 American Journal of Pathology 157:2003-2010).
Accordingly, it is desirable to provide in the field of the present invention, a transgenic animal and cells derived therefrom, which is useful as a model for Alzheimer""s disease and related neurological diseases, and which preferably expresses ACT within the brain of the animal. Such a transgenic animal and cells would be desirable in the study and development of therapeutic approaches toward the treatment and management of such neurological diseases. For example, such transgenic animal and cells would be advantageous in the screening of compounds for the treatment and management of Alzheimer and related neurological diseases. These advantages and more will be apparent to one of ordinary skill in the art upon reading the following disclosure and examples.
It is therefore a feature and advantage of the present invention to provide a transgenic mammal, preferably a mouse, which serves as a model for Alzheimer""s disease and related neurological disorders having a pathology comprising amyloid plaque formation. This transgenic animal carries an exogenous polynucleotide which has a coding sequence functionally equivalent of the DNA sequence of a protease inhibitor such as, for example, human antichymotrypsin (ACT), together with DNA sequences directing its expression, preferably a glial fibrillary acidic protein (GFAP) promoter and 5xe2x80x20 UTR, most preferably modified to remove ATG start codons from its sequence. The mammalian polynucleotide can be in the form of DNA, genomic DNA, cDNA, mRNA and various fragments and portions of the gene sequence encoding ACT. Accordingly, the transgenic mouse exhibits one or more of the symptoms of cognitive memory loss and/or behavioral disturbances, amyloid accumulation, neuronal cell death or synapse loss, formation or aggregation of abnormal protein filaments, or phosphorylation of one or more proteins related to Alzheimer""s disease such as tau. In addition or alternatively, the symptoms can appear as another cellular tissue disorder such as in mouse liver, kidney, spleen, bone marrow or other organs in which the human ACT gene product is expressed.
According to the second embodiment of the invention, animals, primary cell cultures, and cell lines are provided which derive from the parent transgenic animal carrying ACT gene. Accordingly these descendant animals, primary cell cultures and cell lines, which are defined hereinafter as a xe2x80x9cprogenyxe2x80x9d, are either homozygous or heterozygous for ACT allele.
Accordingly, in another embodiment, the transgenic ACT animal expresses one or more additional transgenes of proteins whose expression may be associated with Alzheimer""s disease or related neurological disorders, where the second transgene encodes a normal, mutant, or altered gene encoding, for example, tau-1, apolipoprotein E, APP, presenilin 1, presenilin 2, IL-1 alpha, or IL-1 beta.
As another embodiment of the invention a screening method is provided wherein various test compounds are screened using transgenic animals and/or progeny of the invention. Compounds that are found to have an effect on ACT expression, or to promote or inhibit any of the diverse biochemical effects of ACT expression, are then further tested and used in treatment of Alzheimer""s disease and/or related neurological disorders. In accordance with another aspect of the invention, progeny of the invention can be used as starting points for rational drug design to provide ligands, therapeutic drugs or other types of small chemical molecules. Alternatively, small molecules or other compounds identified by the above-described screening assays can serve as xe2x80x9clead compoundsxe2x80x9d in rational drug design.
There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described below and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.